Studying The Functions Of WLM Family Proteins

Proteins involved in DNA metabolism often engage in transient interactions with DNA.Various agents such as UV light,reactive aldehydes,and chemotherapeutic drugs can permanently and covalently trap DNA-interacting proteins to DNA[1],forming DNA-protein crosslinks(DPCs).DPCs are a severe type of DNA damage,and if left unrepaired,can perturb essential DNA transactions such as DNA replication and transcription[1].Canonical DNA repair pathways such as homologous recombination(HR)and nucleotide excision repair(NER)contribute but do not fully account for the DPC removal processes in the cell[2].Recently,Wss1,a protein possessing protease activity toward DNA-bound proteins,was shown to promote DPC repair in the budding yeast Saccharomyces cerevisiae[3,4].A Wss1-related protein in mammals,SPRTN/DVC1,was found to also participate in DPC removal[5-9].Wss1 belongs to the WLM(Wss1-like metalloprotease family)[10-12].WLM family proteins often harbor protein-protein interaction motifs at the C terminus,including the motifs interacting with Cdc48.Cdc48,which is called p97 or VCP in metazoans,is a critical ATPase involved in various cellular processes by segregating ubiquitinated proteins from complexes or membranes.The functions of Cdc48 are regulated by a diverse group of cofactors,the majority of which contain conserved Cdc48-binding motifs or domains[15].Wssl has two types of Cdc48-interacting motifs called VIM(VCP-interacting motif)and SHP box.The functional relationship between Wssl and Cdc48 remains unclear.It has been reported that Saccharomyces cerevisiae wssl ? mutant is mildly sensitive to a number of DNA-damaging agents,including formaldehyde,UV light,and hydroxyurea[3,13,14].This study found that wss1? mutant is severely sensitive to a nitrofuran compound 3-(5-nitro-2-furyl)acrylic acid(5NFA for short),which can cause DNA damage.Wss1 resists to 5NFA treatment in a manner depending on a catalytic residue in its protease domain and its ability to interact with Cdc48.A spontaneous suppressor screen isolated multiple loss-of-function UBX5 mutations that can rescue the 5NFA sensitivity of wss1?.Interestingly,UBX5 deletion can rescue other phenotypes of wss1? mutant as well.Ubx5 is a known Cdc48 cofactor and uses a UBX domain to interact with Cdc48.Therefore,this study found a suppression genetic interaction between two Cdc48-interacting proteins,Wssl and Ubx5,suggesting that the main function of Wss1 is to antagonize Ubx5.I investigated how Wss1 may antagonize Ubx5.Genetic analysis showed that attenuating the Ubx5-Cdc48 interaction can rescue the phenotypes of wss1? mutant.Affinity purification coupled with mass spectrometry analysis showed that WSS1 deletion resulted in the increased Ubx5-Cdc48 interaction.I predicted that Wssl weakens the interaction between Ubx5 and Cdc48.Supporting this hypothesis,artificially tethering Ubx5 and Cdc48 caused the sensitivity to 5NFA and UV light,mimicking the phenotypes of wss1?.In the fission yeast Schizosaccharomyces pombe,there are two WLM proteins,which were named Wlml and Wlm2.wlm1 single deletion,wlm2 single deletion,and their double deletion are not sensitive to 5NFA and UV light treatment.But wlml wlm2 tdp1 triple mutant is sensitive to the Top1 poison camptothecin(CPT),which can trap Top1 to DNA,forming the Top1cc(Top1 cleavage complex).The function of Wlm1 and Wlm2 in conferring CPT resistance is redundant.A catalytic residue in the protease domain and the Cdc48 binding ability are required for Wlm2 to confer CPT resistance.In summary,this study finds an unexpected suppression genetic interaction between Wssl and Ubx5,and thus reveals a previously unknown aspect of the function of Wssl.In addition,my characterization of fission yeast WLM proteins shed light on the extent of the evolutionary conservation of the functions of WLM family proteins.